Chimaeric phages

ABSTRACT

The invention relates to the field of generating helper phages and phage display libraries for the identification of binding molecules. The invention provide chimaeric phages having a coat comprising a protein mixture. The protein mixture comprises a fusion protein having a proteinaceous molecule fused to a functional form of a phage coat protein and a mutant form of the phage coat protein, wherein the mutant form is impaired in binding to a host cell receptor. The invention further provides new phage collections, novel helper phages and methods and means for producing chimaeric phages, infectious phages and helper phages.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of and claims priority under35 U.S.C. § 365 to PCT/NL02/00391, filed Jun. 14, 2002, designating theUnited States of America, corresponding to PCT International PublicationWO 02/103012 (published in English on Dec. 27, 2002), the contents ofwhich are incorporated herein in its entirety.

TECHNICAL FIELD

The present invention relates generally to biotechnology and moreparticularly to the fields of molecular biology and immunology. Theinvention in particular relates to the field of generating helper phagesand phage display libraries for the identification of binding molecules.

BACKGROUND

An individual needs to have a dynamic immune system that is able toadapt rapidly and respond adequately to potentially harmfulmicroorganisms, and to respond to the exposure of a highly diverse andcontinuously changing environment. Higher organisms have evolvedspecialized molecular mechanisms to ensure the implementation ofclonally-distributed, highly diverse repertoires of antigen-receptormolecules expressed by cells of the immune system: immunoglobulin (Ig)molecules on B lymphocytes and T cell receptors on T lymphocytes. Aprimary repertoire of (generally low affinity) Ig receptors isestablished during B cell differentiation in the bone marrow as a resultof rearrangement of germ line-encoded gene segments. Further refinementof Ig receptor specificity and affinity occurs in peripheral lymphoidorgans where antigen-stimulated B lymphocytes activate a somatichypermutation machinery that specifically targets the immunoglobulinvariable (V) regions. During this process, B cell clones with mutant Igreceptors of higher affinity for the inciting antigen are stimulatedinto clonal proliferation and maturation into antibody-secreting plasmacells (reviewed in Berek and Milstein. 1987).

Recombinant DNA technology has been used to mimic many aspects of theprocesses that govern the generation and selection of natural humanantibody repertoires (reviewed in Winter and Milstein. 1991; Vaughan etal. 1998). The construction of large repertoires of antibody fragments(such as Fab fragments or single chain Fv fragments, scFv's) expressedon the surface of filamentous phage particles and the selection of suchphages by “panning” on antigens has been developed as a versatile andrapid method to obtain antibodies of desired specificities (reviewed inBurton and Barbas. 1994). A subsequent optimization of the affinity ofindividual phage antibodies was achieved by creating mutant antibodyrepertoires of the selected phages and sampled for higher affinitydescendents by selection for binding to antigen under more stringentconditions (reviewed in Hoogenboom. 1994).

M13 and M13-derived phages (sometimes also called viruses) arefilamentous phages that can selectively infect F-pili bearing (F⁺)Escherichia coli (E.coli) cells. The phage genome encodes 11 proteins,while the phage coat itself consists of 5 of these proteins: gene3, -6,-7, -8 and -9 (g3, g6, g7, g8 and g9) proteins that are bound to andthat protect the (circular) single stranded DNA (ssDNA) of the viralgenome. The life cycle of the virus can be subdivided into differentphases.

The g3 protein (g3p) of M13 phages and M13-derivatives comprises threefunctional domains: D1, D2 and D3, linked by two glycine-rich linkers.An alternative nomenclature for g3p domains has also been generallyaccepted, in which D1, D2 and D3 are named “N1,” “N2” and “CT,”respectively. The N-terminal D1/D2 regions interact with the C-terminalD3 region as has been found by Chatellier et al. (1999) using severaldeletion mutants of g3p. Considering that functionality of a D3 domainof the protein is required for assembly of stable phages, a less-, ornon-infectious mutant of the phage coat protein preferably comprises aD3 region of the g3p, or comprises a functional part, derivative and/oranalogue of the D3 region. The D3 domain is thought to bind to DNAinside the viral particle. Loss of the D3 domain functionally results inrare phage-like particles that are very long and very fragile (Pratt etal. 1969; Crissman and Smith. 1984; Rakonjac and Model. 1998). The D1and D2 domain are thought to interact with each other until the phagebinds to the bacteria, while D1/D2 also interact with D3 at certainstages (Chatellier et al. 1999). The linkers present in g3p between D1,D2 and D3 apparently also play a role in infectivity of the phageparticle (Nilsson et al. 2000).

Studies in which a protease cleavage site was introduced between D1 andD2 showed that after cleavage, the phage particle became non-infectious(Kristensen and Winter. 1998). Functional analysis of g3p showed that ofthe g3p N-terminal regions, the D1 domain is essential for infection.Loss of this domain results in phages that cannot infect bacteria(Lubkowski et al. 1998; Nelson et al. 1981; Deng et al. 1999; Riechmannand Holliger. 1997; Holliger and Riechmann. 1997). It has been shownthat the D2 domain interacts with the D1 domain of g3p on the phage(FIG. 1). Due to competition of proteins located on the F-pilus (on F⁺bacteria) that have higher affinity for D2 than for D1, the D1 and D2domain of the g3p dissociate from each other.

The binding of D2 to the F-pilus results in a process that leads toretraction of the F-pilus towards the E.coli cell membrane. Due to thisprocess, the phage particle comes in close contact with the bacterialmembrane. The dissociated D1 domain can interact with bacterial proteinssuch as the TolA receptor, leading to the introduction of the phage DNAinto the E. coli cell (Lubkowski et al. 1999). The fact that removal ofthe D2 domain does not prevent infection, but enables phages to infectE.coli lacking F-pili (Riechmann and Holliger. 1997; Deng et al. 1999)shows that the presence of the D2 domain increases specificity and thatD2 has an important role in preventing F-pili independent infections.The binding of D1 to the specific receptors on the surface of the E.colicell (a feature that is not F⁺-specific) is represented in FIG. 2. Thisprocess triggers the injection of the viral genome into the bacterium(as depicted in FIG. 3).

Although loss of the D2 domain results in the formation of phageparticles that can infect E.coli in a somewhat reduced specific manner,it appears that the level of infections from such a population of phagesis significantly reduced. After infection of an E.coli by a phageparticle, the ssDNA of the virus becomes double stranded due to theaction of a number of bacterial enzymes. The double stranded phagegenome serves as a template for the transcription and translation of all11 genes located on the phage genome. Besides these protein-encodingregions, the phage genome contains an intergenic region: the F1-originof replication initiation (F1-ORI). The DNA sequence of this F1-ORI canbe divided in 2 separate subregions. One subregion is responsible forthe initiation and termination of the synthesis of ssDNA via theso-called ‘rolling circle mechanism’ and the other subregion isresponsible for the packaging initiation of the formed circular ssDNAleading to the formation and release of new virus particles.

It has been shown that polypeptides, such as stretches of amino acids,protein parts or even entire proteins can be added by means of moleculargenetics to the terminal ends of a number of particle coat proteins,without disturbing the functionality of these proteins in the phage lifecycle (Smith. 1985; Cwirla et al. 1990; Devlin et al. 1990; Bass et al.1990; Felici et al. 1993; Luzzago et al. 1993).

This feature enables investigators to display peptides or proteins onphages, resulting in the generation of peptide- or protein expressionphage display libraries. One of the proteins that has been used to fusewith polypeptides for phage display purposes, is the g3 protein (g3p),which is a coat protein that is required for an efficient and effectiveinfectivity and subsequent entry of the viral genome into the E. colicell.

For the production of phages that display polypeptides fused to the g3pcoat protein, investigators introduced a plasmid together with the phagegenome in E.coli cells. This plasmid contains an active promoterupstream of an in-frame fusion between the g3 encoding gene and a geneof interest (X) encoding, for instance, polypeptides such as proteinssuch as antibodies or fragments such as Fab fragments or scFv's. Theintroduction of this plasmid together with the genome of the helperphage in an E.coli cell results in the generation of phages that containon their coat either the wild type g3p from the viral genome, the fusionproduct g3p-X from the plasmid or a mixture of the two, since one phageparticle carries five g3p's on its surface. The process of g3p or g3p-Xincorporation is generally random.

The presence of an F1-ORI sequence in the g3p-X expression vector(plasmid) misleads the phage synthesis machinery in such a way that twotypes of circular ssDNA are formed: one is derived from the genome ofthe phage and the other is derived from the expression vector. Duringthe synthesis of new phages, the machinery is unable to distinguish thedifference between these two forms of ssDNA resulting in the synthesisof a mixed population of phages, one part containing the phage genomeand one part harboring the vector DNA. Due to these processes, themixture contains at least some phages in which the phenotypicinformation on the outside (the g3p-X fusion protein) is conservedwithin the genotypic information inside the particle (the g3p-Xexpression vector). An infectious wild type phage and a phage carrying afusion protein attached to g3p are depicted in FIG. 4. The art teachesthat there are several problems that concern the use of these basicset-ups.

The high level of genotypic wild type phages in phage populations grownin bacteria that contain both the phage genome and the expression vectorcompelled investigators to design mutant F1-ORI sequences in M13genomes. Such mutant M13-strains are less effective in incorporatingtheir genome in phage particles during phage assembly, resulting in anincreased percentage of phages containing vector sequences whenco-expressed. These mutant phages, such as the commercially availablestrains R408, VCSM13 and M13KO7, are called “helper phages.” The genomeof these helper phages may contain genes required to assemble new(helper-) phages in E.coli and to subsequently infect new F-piliexpressing E.coli. Both VCSM13 and M13K07 were provided with an originof replication initiation (ORI) of the P15A type that results in themultiplication of the viral genome in E.coli. Moreover, the ORIintroduction ensures that after cell division the old and newly formedE.coli contains at least one copy of the viral genome.

It was suggested and finally proven by several investigators that theintroduction of plasmids containing a g3p-scFv fusion product togetherwith the genome of the helper phages in E.coli cells results inapproximately 99% of newly formed phages that harbor the g3p-scFv fusionprotein expression plasmid, but nevertheless lack the g3p-scFv fusion onits surface (Beekwilder et al. 1999). The absence of g3p-X is asignificant disadvantage in the use of display libraries for theidentification of specific proteins or peptides such as scFv's that bindto a target of interest (such as tumor antigens). It implies that in thecase of phage display libraries, at least a 100-fold excess of producedphages must be used in an experiment in order to perform a selectionwith all possible fusion proteins present.

The art teaches that this overload of relatively useless phages in anexperiment leads to (too) many false positives. For instance, at least10¹² phages should be added to a panning experiment in order to have 1copy of each possible fusion present in the experiment, since such alibrary contains approximately 10¹⁰ different g3p-scFv fusions (1%). Thephages in this approximate 1% express generally only 1 g3p-scFv fusionon their coat together with four normal g3p's (no fusions), while therest of the helper phages (approximately 99%) express five g3p's and nog3p-scFv fusions. To ensure, theoretically, the presence of 100 copiesof each separate fusion protein in a panning experiment, one needs touse approximately 10¹⁴ phages in such an experiment. Persons skilled inthe art generally attempt to use an excess of at least 100-fold of eachsingle unique fusion protein, to ensure the presence of sufficientnumbers of each separate fusion and not to lose relevant binders tooquickly in first panning rounds. That number of phages (10¹⁴) is more orless the maximum of phage particles that a milliliter (ml) can hold. Theviscosity of such a solution is extremely high and therefore relativelyuseless. Especially when ELISA panning strategies are used (in which thevolume of one well is only 200 μl) such libraries cannot be used.

In addition to these problems, it is assumed that, generally, dependingon the antigen and the stringency of washing procedures, an average of 1in every 10⁷ phages will bind to the antigen due to a-specific binding.Thus, for the application of 10¹² scFv expressing input phages (1%) to apanning procedure, one has to add approximately 10¹⁴ phages (99% ofwhich do not express a scFv fragment). It is generally assumed that fromthese 10¹² phages, approximately 10⁴ particles might be putativelyinteresting phages. However, depending on the washing conditions, thenumber of calculated background phages that are normally found by usinglibraries present in the art after one round of panning, wasapproximately 10⁶-10⁷ while only a few of these phages appear to berelevant binders. This is one of the most significant problemsrecognized in the art: too many background phages show up as initialbinders in the phage mix after the first round of panning, while only afew significant and interesting binders are present in this mix. Thus,the absolute number of isolated phages after one round of panning isclearly too high (10⁶-10⁷). Moreover, in subsequent rounds of panning,non-specific background phages remain present. In libraries used in theart, most of these non-specific binders will amplify on bacteria that,upon amplification, continue to in a second round of panning. Therefore,the art teaches that the background level of a-specifically bindingphages and the total number of phages per ml in these types of librariesis unacceptably high and remains high during subsequent rounds ofpanning.

A possibility that was suggested by investigators in the art as asolution to the problem of obtaining too many background phages thatlack a g3p-X fusion was to remove the g3p-encoding gene entirely fromthe helper phage genome. In principle, this system ensures that duringphage synthesis in an E.coli cell (that received the g3-less phagegenome and a g3p-X fusion protein expression vector), only g3p-Xproteins are incorporated in the newly formed phage coat. By doing so,each synthesized phage will express five copies of the g3p-X fusionproduct and hardly any phages are synthesized that express the g3p aloneor that express less than five g3p-X fusions. R408-d3 and M13MDΔD3 aretwo examples of g3-minus helper phages (Dueñias and Borrebaeck. 1995;Rakonjac et al. 1997). Because the genome of these phages is not capableof supporting g3p synthesis, phage particles that carry less than fiveg3p-X fusion proteins can hardly be formed; or, if formed, are found tobe non-infectious due to instability, since the art teaches that fiveg3p's are necessary to ensure a stable phage particle.

To produce helper phages that do not contain the g3 gene, but that arenevertheless infectious and that can be used to generate libraries ofphages that carry five g3p-X fusion proteins, and that lack phages withless than five g3p-X fusions, it was recognized in the art that anexternal source for g3p was required. Such a source can be a vectorwithout F1-ORI but that nevertheless contains an active promoterupstream of the full open reading frame (ORF) of g3. One major problemthat is recognized by persons skilled in the art is that after thegeneration step of producing newly formed helper phages lacking a g3gene, the yield is dramatically low. In fact, the yield of all describedsystems is below 10¹⁰ phages per liter, meaning that for a library of10¹⁰ individual clones, at least 100 liters of helper phage culture arenecessary (NB: the helper phages need to be purified) in order to growthe library once. The art, thus, teaches that phage libraries generatedwith such low titers of helper phages are not useful for phage displaypurposes and that these libraries cannot be used for panningexperiments. One method for complementation of g3p deletion phages waspresented recently, in which wild-type g3p was provided by a nucleicacid encoding the wild-type g3p, wherein the nucleic acid was stablyincorporated into the host cell genome (Rondot et al. 2001).

Phages that express deleted g3p's fused to heterologous proteins havebeen generated. For the construction of most conventional Fab librariesand some scFv phage display libraries, the D1 domain and parts of the D2domain were removed to ensure a shorter fusion protein, which wasconsidered in the art as a product that could be translated easier thana full length g3p linked to a full length Fab fragment. The shorter g3ppart would not prevent the generation of a viable and useful helperphage. Of course, such phages still depend on full length g3p's that arepresent on their surface next to the deleted g3p fusion with the Fabfragment for functional infectivity of E.coli cells. Also, phages thatexpress deleted g3p's fused to ligand-binding proteins have beengenerated that depend on their infectious abilities on antigens thatwere fused to the parts of g3p that were missing from the non-infectiousphage particle (Krebber et al. 1997; Spada et al. 1997). These particlesdepend for their infectivity on an interaction between theligand-binding protein, such as an antibody or a fragment thereof andtheir respective ligand (or antigen). However, thisinteraction-dependency reduces the efficiency of infection, due toelimination of a direct linkage between the g3p domains, and a generalinhibitory effect of the soluble N-terminal part of g3p coupled to theantigen.

The g3-minus helper phages R408-d3 and M13MDΔD3 mentioned above lack abacterial ORI and a selection marker in their genome. The absence of aselection marker in the g3-minus genomes has a significant effect on theproduction scale of helper phages, because it results in an overgrowthof bacteria that do not contain the helper phage genome. It is knownthat bacteria grow slower when infected with the helper phage or virus.Therefore, bacteria that lack the phage genome quickly overgrow theother bacteria that do contain the genome. Another effect of the lack ofan ORI or a selection marker is that g3-minus phage genomes cannot bekept in dividing bacteria during the production and expansion of phagedisplay libraries. This is a very important negative feature becauseovergrowth of bacteria that lost the phage genome or that never receivedone, appear to have a growth advantage over bacteria that do contain thephage genome. In addition, such ‘empty’ bacteria are not capable ofproducing any phage and as a result, the phage display vectors includingfusion protein fragments in such helper phages lacking-bacteria are lostpermanently.

As mentioned, the g3p's are thought to be essential for the assembly ofstable M13-like phages and because of their crucial role in infection,g3p's should be provided otherwise when g3-minus helper phages are to begenerated. There is a prejudice in the art against making phage displaylibraries that lack g3p's because phages lacking g3p's are not stable.Rakonjac et al. (1997) constructed a VCSM13 g3-minus helper phage inparallel to a R408 g3-minus helper phage and used helper plasmids witheither the psp or the lac promoter upstream of a full length g3 sequenceto substitute g3 during helper phage synthesis (Model et al. 1997).However, the art teaches that the lac promoter has the disadvantage thatit cannot be shut off completely, even not in the presence of highconcentrations of glucose (3-5%) in the medium (Rakonjac and Model.1998).

An additional problem that is well known in the art is that even verylow levels of g3p in E.coli can block infection of M13-like phages.Moreover, it has been shown that co-encapsidation of plasmids togetherwith the phage genome can occur (Russel and Model. 1989; Krebber et al.1995; Rakonjac et al. 1997). If co-encapsidation occurs with the lacdriven helper plasmid, it will compete with the lac driven vectors usedin the phage display resulting in the efficient production of infectiousphage particles that will not contain the g3p-X fusion product.Together, the art thus teaches that the lac promoter is not the bestcandidate promoter in the helper plasmid system. The psp promoter hasthe advantage to be relatively silent in E.coli until infection(Rakonjac et al. 1997). Upon M13-class phage infection, the psp promoterbecomes activated and the helper plasmid will produce g3 proteins.However, the disadvantage of this promoter is that the level of RNAproduction cannot be regulated with external factors, but has to beregulated by either mutating (and change the activity of) the promotor,changing the ribosomal binding site (RBS) or other elements thatinfluence the promotor activity. To figure out the ideal level ofpromotor activity in a specific E.coli strain can be time consuming andneeds to be optimized for each E.coli strain separately. The art alsoteaches that the psp promotor system is not very attractive forlarge-scale helper phage production due to the inflexibility of E.colistrains, the time consuming optimization and the significant low levelof helper phage production.

A significant problematic feature helper phage systems described is theoccurrence of unwanted recombination events between the helper genomeand the (helper-) plasmids. The problem that confronts investigators inthe art is the fact that the g3 DNA sequences in the helper phages arehomologous to the g3 sequences in the phage display vector and/or thehelper phage plasmid. This results, in many cases, in recombinationbetween the two DNA strains and therefore loss of functionality of thelibrary as a whole.

SUMMARY OF THE INVENTION

The current invention provides chimaeric phages, novel helper phages,libraries comprising the chimaeric phages and methods and means toproduce the chimaeric phages and the helper phages.

In one embodiment, the invention provides a chimaeric phage having acoat comprising a mixture of proteins, the mixture comprising a fusionprotein, wherein a proteinaceous molecule is fused to a functional formof a phage coat protein. The mixture further comprises a mutant form ofthe phage coat protein, the mutant form being impaired in binding to ahost cell receptor. The invention also provides a chimaeric phage havinga coat comprising a mixture of proteins, the mixture comprising a fusionprotein, wherein a proteinaceous molecule is fused to a phage coatprotein, or to a fragment or derivative thereof The fusion protein isfunctional so as to render the chimaeric phage infectious, the mixturefurther comprises a mutant form of the phage coat protein, the mutantform being impaired in binding to a host cell receptor.

In another embodiment, the invention provides a helper phage comprisinga nucleic acid encoding phage proteins or functional equivalents thereofthat are essential for the assembly of the helper phage. The nucleicacid further encodes a mutant form of a phage coat protein, the mutantform being impaired in binding to a host cell receptor, and wherein thehelper phage does not comprise nucleic acid encoding a functional formof the phage coat protein.

In yet another embodiment, the invention further provides methods andmeans for producing phage particles, chimaeric phages, infectious phagesand helper phages. The invention also provides phage collections, suchas phage display libraries comprising chimaeric phages and/or infectiousphages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the g3 protein (g3p) present in thecoat of M13 phages. The D3 domain of g3p is attached to the singlestranded DNA inside the particle via the g8 protein (g8p), while the D1and D2 domains interact with each other outside the particle and can beused for fusion with for example scFv.

FIG. 2. Schematic representation of the interaction between the D2domain of g3p with the F-pilus on the surface of E.coli, with asubsequent interaction of the D1 domain with other components of thebacterial surface.

FIG. 3. Schematic representation of the interaction of the D2 domain ofg3p with the F-pilus (left) and the D1 domain of g3p with the TolAreceptor (see FIG. 2) and the subsequent entry (right) of the phagegenome into the cytoplasm of the bacterium.

FIG. 4. Schematic representation of a wild type phage expressing fiveg3p's on its infectious end (left) and a recombinant phage expressingfour wild type g3p's and one g3p-X fusion protein on its infectious end(right). The recombinant phage also harbors the genetic information ofthe fusion protein present on the surface.

FIG. 5. Schematic representation of the pBAD/gIII-g3 helper vectorharboring the full-length g3 gene under the control of the AraC/BADpromoter and further harboring an ampicillin resistance and a ColE1origin of replication (ORI).

FIGS. 6A-6D. (FIG. 6A) Schematic representation of the VCSM13 helperphage genome. The 11 genes are indicated, as well as the kanamycinresistance gene (Kan R), the packaging signal (PS) and the large andsmall fragments of the original intergenic region (IG). (FIG. 6B)Sequence of the VCSM13 genome(SEQ ID NO:15) shown in FIG. 6A. Thetranslation of the g3 gene (SEQ ID NO: 16) is given in one letter code.(FIG. 6C) Schematic representation of the VCSM13-derived g3-minus helperphage genome deleted for the open reading frame (ORF) of the g3 gene.(FIG. 6D) Sequence (SEQ ID NO:17) of the part of the helper phage genomethat surrounds the position of the g3 deletion depicted in FIG. 6C. TheHindIII site at position 3431 is underlined, followed by the 6 codons inbold face upstream of the TAA stop codon.

FIGS. 7A-7 b. (FIG. 7A) Schematic representation of the VCSM13-derivedD3 helper phage genome deleted for the D1 and D2 domains of the g3 gene.The D3 part of the g3 gene encodes the carboxy-terminal part of the g3protein enabling the generation of stable, but essentiallynon-infectious helper phages. The D3 helper phage genome is identical tothe CT helper phage genome. (FIG. 7B) Sequence (SEQ ID NO: 18) of thesequence listing incorporated herein of the D3 domain of g3 present inthe nucleic acid shown in FIG. 7A. The BamHI site at position 3488 aswell as the GTG start en TAA stop codon are underlined.

FIG. 8. Codon usage adaptation in the g3 gene and the D3 domain toprevent homologous recombination during helper phage production andduring phage display library amplification. The left panel shows theobtained one-letter coded amino acids and the right panel shows theoptimal codons.

FIG. 9. Schematic representation of the pUC-g3 helper plasmid harboringthe full length g3 gene under the control of the lac promoter andfurther harboring an ampicillin resistance gene (Amp R) and a ColE1origin of replication (ORI).

FIG. 10. Schematic representation of the VCSM13-derived N2CT helperphage genome deleted for the N1 (D1) domain of the g3 gene that confersinfectivity to the phage.

FIG. 11. Schematic representation of the VCSM13-derived p3-minus helperphage genome expressing the g3 leader followed by only seven aminoacids. At the DNA level, a large part of the N2 (D2) domain and theentire CT (D3) domain are still present. The former C-terminal region ofgene III is present, but does not encode functional protein throughframe shift and therefore includes many in-frame stop codons.

DETAILED DESCRIPTION

The present invention provides a chimaeric phage having a coatcomprising a mixture of proteins, the mixture comprising a fusionprotein, wherein a proteinaceous molecule is fused to a functional formof a phage coat protein. The mixture further comprises a mutant form ofthe phage coat protein, wherein the mutant form is characterized in thata phage comprising no wild type phage coat protein from which the mutantform is derived and having a coat comprising the mutant form and nocopies of the functional form, is less infectious than a phagecomprising no wild type phage coat protein from which the mutant form isderived and having a coat comprising the mutant form and at least onecopy of the functional form.

In one embodiment of the invention, the mutant form is characterized inthat a phage comprising no wild type phage coat protein from which themutant form is derived and carrying the mutant form and no copies of thefusion protein is less infectious than a phage comprising no wild typephage coat protein from which the mutant form is derived and carrying inaddition to the mutant form, at least one copy of the fusion protein.

Preferably, the mutant form is characterized in that a phage comprisingno wild type phage coat protein from which the mutant form is derivedand carrying the mutant form and no copies of the fusion protein or thefunctional form is non-infectious. More preferably, the mutant form isfurther characterized in that a phage having a coat comprising themutant form in the presence or absence of copies of the functional form,is stable. Stable as used herein means that the part of g3p that isstill present in the mutant form (and that is also present in thefunctional form) ensures features such as DNA binding and rigidity ofthe phage, but does not contribute to infectiousness of the phage as dothe domains in the functional form that are not present in the mutantform.

The invention also provides an infectious phage containing at least onecopy of a mutant form of a phage coat protein, wherein the mutant formhas lost the ability to mediate infection of a natural host by theinfectious phage.

In a preferred embodiment, the phage coat protein is the g3 protein(g3p) present in the coat of phages such as M13 and R408. Morepreferably, the mutant form comprises a mutation in the D1 and/or the D2region of g3p.

In another preferred aspect of the invention, the chimaeric phage and/orthe infectious phage are part of a phage collection such as a phagedisplay library. In a more preferred aspect of the invention, such aphage collection consists essentially of chimaeric phages or infectiousphages provided by the invention. Also preferred are chimaeric phages orinfectious phages according to the invention that comprise bindingmoieties, such as antibodies or fragments thereof as part of the fusionprotein.

In another embodiment, the invention provides a method for producing aphage particle comprising the steps of: providing a host cell with afirst nucleic acid encoding a fusion protein, the fusion proteincomprising a proteinaceous molecule fused to a functional form of aphage coat protein; providing the host cell with a second nucleic acidencoding a mutant form of the phage coat protein, the mutant form beingcharacterized in that a phage comprising no wild type phage coat proteinfrom which the mutant form is derived and having a coat comprising themutant form and no copies of the functional form is less infectious thana phage comprising no wild type phage coat protein from which the mutantform is derived and having a coat comprising at least one copy of thefunctional form. The host cell further comprises an additional nucleicacid sequence encoding at least all other proteins or functionalequivalents thereof that are essential for the assembly of the phageparticle in the host cell and the method further comprises culturing thehost cell to allow assembly of the phage particle.

The invention provides a method for producing a phage particle, themethod comprising the steps of: providing a host cell with a firstnucleic acid encoding a fusion protein, the fusion protein comprising aproteinaceous molecule fused to a functional form of a phage coatprotein; providing the host cell with a second nucleic acid encoding amutant form of the phage coat protein, the mutant form being impaired inbinding to a host cell receptor; and culturing the host cell to allowassembly of the phage particle.

Preferably, these methods according to the invention for producing aphage particle are applied for producing the chimaeric phage and/or theinfectious phage. More preferably, the method is applied for producing aphage particle such as a chimaeric phage or an infectious phage providedby the invention that comprise nucleic acid encoding the mutant formunder the control of a controllable promoter such as the AraC/BADpromoter, the lac promoter or the psp promoter.

In another embodiment, the invention provides a helper phage comprisingnucleic acid encoding phage proteins or functional equivalents thereofthat are essential for the assembly of the helper phage. The nucleicacid further encodes a mutant form of a phage coat protein, wherein themutant form is characterized in that a phage comprising no wild typephage coat protein from which the mutant form is derived and having acoat comprising the mutant form and no copies of a functional form ofthe phage coat protein, is less infectious than a phage comprising nowild type phage coat protein from which the mutant form is derived andhas a coat comprising at least one copy of the functional form, whereinthe functional form is characterized in that it renders a phage particlecarrying the functional form in its coat infectious, and wherein thehelper phage does not comprise expressible nucleic acid encoding thefunctional form. The invention provides a helper phage comprisingnucleic acid encoding phage proteins or functional equivalents thereofthat are essential for the assembly of the helper phage, the nucleicacid further encodes a mutant form of a phage coat protein, the mutantform being impaired in binding to a host cell receptor, and wherein thehelper phage does not comprise nucleic acid encoding a functional formof the phage coat protein.

In yet another embodiment, the invention provides a method for producinga helper phage comprising the steps of: providing a host cell with afirst nucleic acid encoding a functional form of a phage coat protein;providing the host cell with a second nucleic acid encoding a mutantform of the phage coat protein, wherein the mutant form is characterizedin that a phage comprising no wild type phage coat protein from whichthe mutant form is derived. The helper phage has a coat comprising themutant form and is less infectious than a phage comprising no wild typephage coat protein from which the mutant form is derived and has a coatcomprising at least one copy of the functional form. The host cellcomprises an additional nucleic acid sequence encoding at least allother proteins or functional equivalents thereof that are essential forthe assembly of the helper phage in the host cell. The method furtherincludes culturing the host cell to allow assembly of the helper phage.

The invention also provides a method for producing a helper phagecomprising the steps of: providing a host cell with a first nucleic acidencoding a functional form of a phage coat protein; providing the hostcell with a second nucleic acid encoding a mutant form of a phage coatprotein, the mutant form being impaired in binding to a host cellreceptor. The host cell comprises an additional nucleic acid sequenceencoding at least all other proteins or functional equivalents thereofthat are essential for the assembly of the helper phage in the hostcell. The method further includes culturing the host cell to allowassembly of the helper phage.

In another aspect, the invention provides methods and means forproducing a phage particle such as a chimaeric phage, an infectiousphage or a helper phage according to the invention, wherein separatenucleic acids encoding either (1) a functional form of the phage coatprotein alone or fused to a proteinaceous molecule or (2) a mutant formof the phage coat protein. The chimaeric phage, the infections phage orthe helper phage each comprise codons in the overlapping regions betweenthe protein encoding parts, that essentially do not render a homologousrecombination event between the separate nucleic acids. In a preferredaspect the separate nucleic acids each comprise non-interfering originsof replication and unique selection markers.

In another embodiment, the invention provides a method for theenrichment of a first binding pair member in a repertoire of firstbinding pair members selected from the group consisting of; an antibody,an antibody fragment, a single chain Fv fragment, a Fab fragment, avariable region, a CDR region, an immunoglobulin or a functional partthereof. The first binding pair member is specific for a second bindingpair member. The method comprises the steps of: contacting a phagecollection comprising chimaeric or infectious phages according to theinvention with material comprising the second binding pair member underconditions allowing specific binding; removing non-specific binders; andrecovering specific binders. The specific binders comprise the firstbinding pair member. The material may comprise second binding pairmembers such as purified proteins, recombinant proteins and/or proteinspresent in or on cells.

In a preferred embodiment, the invention provides a method for theenrichment of a first binding pair member that comprises the steps of:recovering from a phage a DNA sequence encoding the first specificbinding pair member; sub-cloning the DNA sequence in a suitableexpression vector; expressing the DNA sequence in a suitable host; andculturing the suitable host under conditions whereby the first specificbinding pair member is produced. A suitable expression vector may be aplasmid vector comprising an active promoter that regulates theexpression of the first specific binding pair member in suitable hostsincluding eukaryotic cells, such as, yeast cells or mammalian cells.

In another aspect, the invention provides a nucleic acid moleculecomprising a sequence encoding a mutant form of a phage coat protein.The mutant form is characterized in that a phage, comprising no wildtype phage coat protein from which the mutant form is derived and havinga coat comprising the mutant form and no functional form of the phagecoat protein, is less infectious than a phage comprising no wild typephage coat protein from which the mutant form is derived and having acoat comprising the mutant form and at least one copy of the functionalform of the phage coat protein. The functional form is characterized inthat it renders a phage carrying the functional form in its coatinfectious. The nucleic acid molecule may furthermore comprise allrelevant nucleic acid encoding proteins that are required for assemblyof a phage in a host cell.

The invention provides a chimaeric phage having a coat comprising amixture of proteins, the mixture comprising a fusion protein, wherein aproteinaceous molecule is fused to a functional form of a phage coatprotein. The mixture further comprises a mutant form of the phage coatprotein, wherein the mutant form is characterized in that a phagecomprising no wild type phage coat protein from which the mutant form isderived and having a coat comprising a mutant form of the phage coatprotein and no copy or copies of the functional form of the phage coatprotein, is less infectious than a phage comprising no wild type phagecoat protein from which the mutant form is derived and having a coatcomprising at least one functional form of the phage coat protein.

“Functional form” as used herein refers to a phage coat protein thatcontributes significantly to the infectiousness of the particle to whichit is attached. The phage coat protein itself is not infectious, but thefunctional form renders the phage particle to which it is attachedinfectious. Besides the contribution to infectivity of the phageparticle, the phage coat protein also sustains other functions, such asstabilization of the phage particle.

A “mutant form” of a phage coat protein according to the invention mayrender the phage particle less infectious or non-infectious, but shouldstill sustain other functions of the phage coat protein such asstabilization of the phage. A phage that comprises no wild type phagecoat protein from which the mutant form is derived or originates andcomprises no functional forms of the phage coat protein, but doescontain only mutant forms of the phage coat protein is less infectiousthan a phage that comprises no wild type phage coat protein from whichthe mutant form is derived and comprises one or more functional forms ofthe phage coat protein next to the mutant forms of the phage coatprotein in its coat. “Less infectious” as used herein may also meannon-infectious.

Although a chimaeric phage of the invention comprises at least one copyof the mutant form of the phage coat protein in its coat, the chimaericphage has infectious capability because it also comprises at least onefunctional form of the phage coat protein in its coat.

A “functional form of a phage coat protein” as used herein also means apart, derivative and/or an analogue thereof that still harborsfunctionality in rendering the phage infectious to which it is attached.

“Mutant form of a phage coat protein” as used herein also means a part,a derivative and/or an analogue of the mutant form, wherein the mutantform is characterized in that a phage, comprising no wild type phagecoat protein from which the mutant form is derived and having a coatcomprising only mutant forms of the phage coat protein or parts,derivatives and/or analogues thereof, is less- or non-infectious ascompared to a phage comprising no wild type phage coat protein fromwhich the mutant form is derived and having a coat comprising at leastone functional form of the phage coat protein.

In a preferred embodiment, the phage coat protein is the g3 protein(g3p) that in a wild type or functional form renders a phage to which itis attached, infectious. As is outlined in FIGS. 2 and 3, certain partsof the g3 protein are involved in the recognition of host cellreceptors.

A mutant form is “impaired” in receptor binding if an alteration in theg3 protein renders the receptor to be recognized and bound by the g3protein, or parts thereof, to a lower extent than if no alteration ispresent. Therefore, “impaired” as used herein means that the coatprotein, such as g3p, binds the host cell receptor less efficiently, orthat g3p has completely lost the ability to bind and/or recognize thehost cell receptor.

In a preferred embodiment, the mutant form of the phage coat proteincomprises an alteration in the g3 protein consisting of a mutation ineither the D1 region, the D2 region or both.

An “alteration” or “mutation” as used herein means one or multiple pointmutations, stretches of mutations, deletions, substitutions,replacements and/or swapping of parts. In a more preferred embodiment,the alteration in the g3 protein is a deletion of substantially all ofthe D1 and/or the D2 region. The alteration may also mean a substitutionof the deleted g3 protein part by a protein or a peptide notcontributing to the infectivity of the helper phage, the chimaericphage, the infectious phage or the phage particle.

In a more preferred embodiment of the invention, a chimaeric phage or aninfectious phage according to the invention comprises a nucleic acidencoding a fusion protein, wherein a proteinaceous molecule is fused toa functional form of the phage coat protein. The chimaeric phage of theinvention comprises a M13, M13K07, VCSM13 or a R408 strain or a mutant,derivative or analogue strain derived from either one of these strains.A proteinaceous molecule according to the invention is fused to thefunctional form of the phage coat protein and comprises a protein, suchas a ligand-binding moiety or an immunoglobulin (such as an antibody). Aproteinaceous molecule may also mean a peptide, such as a random stretchof amino acids or a non-random stretch of amino acids such as anantibody fragment or derivatives thereof (Fab fragment, a single chainFv fragment (scFv), a variable region or a CDR region). A proteinaceousmolecule can also mean first specific binding pair member, fusionsbetween different kinds of (fragments of) proteins and/or fusionsbetween (fragments of) proteins and (random and non-random) peptides,such as antibody-recognized tags.

A chimaeric phage of the invention relies on the functional form of thephage coat protein g3p and on the presence of part(s) of the phage coatprotein that contribute to the infectivity of the phage for infection.The mutant form of the phage coat protein is mutated in the part(s) ofthe phage coat protein that render the phage infectious. The mutation isexemplified in, but not limited to, deletions, residue- or fragmentsubstitutions, swaps and/or replacements by other protein fragmentsrendering it less infectious. The protein fragments may or may not berelated to phage coat proteins or fragments thereof, and are essentiallynot capable of inducing infection of the phage particle into a hostcell.

In another embodiment, the invention provides a phage collectioncomprising a chimaeric phage or an infectious phage according to theinvention. Phages of the present invention are particularly useful forthe generation of phage display libraries. Therefore, in a preferredembodiment, the phage collection is a phage display library. In a morepreferred embodiment, the phage collection consists essentially ofchimaeric phages or of infectious phages of the invention. Aproteinaceous molecule, such as (random or not random) stretches ofamino acids, peptides, protein parts or even entire proteins can befused to the phage coat proteins and can form a first specific bindingpair member. This fusion is typically done at the terminal ends of thecoat protein, wherein the additions typically do not affect the functionof the phage coat protein. Moreover, it also often does not interferewith the function of the added moiety. Thus, it is possible to generatelibraries that can be used to locate and clone specific bindingmolecules. Such libraries can comprise peptides or larger molecules.Preferably, the larger molecules comprise a protein, such as an antibodyor a functional part, derivative and/or analogue thereof, such as fulllength heavy and/or light chains from an immunoglobulin molecule, orfragments of immunoglobulins such as Fab fragments, single chain Fv(scFv) fragments, CDR regions, sole variable regions and/or combinationsof the above.

In another aspect, the invention provides a method for making a phageparticle which comprises the steps of providing a host cell with a firstnucleic acid encoding a fusion protein. The fusion protein comprises aproteinaceous molecule fused to a functional form of a phage coatprotein or to a functional part, derivative and/or analogue of the phagecoat protein. The method further includes providing the host cell with asecond nucleic acid encoding a mutant form of the phage coat protein,the mutant form being characterized as described above. For instance,less infectious may also mean non-infectious. The method also includesculturing the cell to allow assembly of the phage, the host cellotherwise or additionally comprising nucleic acid encoding at least allessential proteins, or functional equivalents of the essential proteinsfor the assembly of the phage particle.

In a preferred embodiment, the invention provides a method wherein thenucleic acid encoding at least all other proteins or functionalequivalents thereof that are essential for the assembly of the phageparticle in the host cell is comprised by a helper phage and the helperphage is used to deliver the nucleic acid to the host cell. In a morepreferred embodiment, the nucleic acid that is delivered by the helperphage also comprises the second nucleic acid encoding the mutant form ofthe phage coat protein. In an even more preferred embodiment, the firstand the second nucleic acids are separate nucleic acids that each maycomprise separate unique selection markers to ensure that the host cellcomprises at least one copy of each separate nucleic acid. The first andsecond nucleic acids each comprise separate unique origins ofreplication to ensure no interferences during replication.

In another aspect of the invention, the number of possible homologousrecombination events between overlapping stretches of nucleic acidsequences between the separate nucleic acids is reduced due to the useof different codons within each nucleic acid.

Using a method of the invention, it is possible to generate phageparticles that comprise at least two variants derived from the same coatprotein. The present invention provides novel phage particles (chimaericphages, infectious phages and helper phages) in which the relativenumber of the g3p variants (either fused to a heterologous moiety ornot) present in the coat can vary. Typically, one wants to influence therelative amount of the various variants in the phage coat. To that end,it is preferred that expression of the fusion protein and/or expressionof the mutant form of the phage coat protein is regulatable(controllable). Preferably, this is achieved by regulating theexpression of the gene encoding the phage coat protein at atranscriptional level.

Thus, preferably, expression of the fusion protein and/or the mutantform of the phage coat protein is under the control of a promoter thatis well controlled. Examples of such promoters are the lac promoter, thepsp promoter and the AraC/BAD promoter, the latter being influenced bythe concentration of glucose or arabinose in the medium.

Particularly advantageous is the AraC/BAD promoter. The AraC/BADpromoter is preferred because it is a promoter that is controlled in avery tight manner. This promoter is for all practical purposes silent inthe presence of glucose and only slightly leaky in the absence ofglucose. This means that a low concentration or absence of glucoserenders the promoter active, resulting in up-regulation of the gene ofinterest that is under the control of the promoter. If, for example, thedeletion mutant of g3 (lacking the D1 and D2 infectious regions) isunder the control of such a promoter, the relative number of deletionmutants, as compared to full length (or at least functional) g3-fusionproteins, would be low when the glucose concentration is relativelyhigh. In principle, this system results in a tight regulation of thenumber of deletion mutants and functional coat proteins such as g3 onthe coat of a phage particle, and therefore the percentage of such coatproteins and deletion mutants can be regulated.

In addition, the activity of the AraC/BAD promoter can be regulated verytightly by the addition of arabinose to the medium. The concentrationarabinose used determines the level of protein expressed in E. colicells. Therefore, optimal regulation of phage coat protein content isaccomplished by using this AraC/BAD promoter and by altering theculturing conditions of the host cell. The use of a promoter that isdependent on arabinose, such as the AraC/BAD promoter, instead of IPTG,such as the lac-operon, prevents possible problems that will occur dueto co-encapsidation of the helper plasmid in viral particles duringhelper phage synthesis.

As described above, it has been shown that co-encapsidation of plasmidstogether with the phage genome does occur (Russel and Model. 1989;Krebber et al. 1995; Rakonjac et al. 1997). If co-encapsidation occurswith a lac driven helper plasmid, it may compete with the lac drivenvectors, generally used for the phage display, resulting in theproduction of infectious phage particles that will not contain the g3p-Xfusion product. This problem most likely does not occur when theAraC/BAD promoter is used. However, in the studies presented by thepresent invention, such competition problems with the lac promoter werenot observed. Therefore, other regulatable promoters such as the psp andlac promoter can be used to generate phages according to the invention.It is therefore also a part of the invention to use such promoters as analternative to the AraC/BAD promoter.

A method for the production of a phage particle is preferably used toproduce a chimaeric phage according to the invention. The host can beprovided with nucleic acid encoding a phage protein in any suitable way.Preferably, however, the host is provided with a helper phage accordingto the invention.

A helper phage according to the invention comprises nucleic acidencoding other phage proteins or functional equivalents thereof that areessential for the assembly of the helper phage, the nucleic acid furtherencoding a mutant form of a phage coat protein. The mutant form ischaracterized in that a phage comprising no wild type phage coat proteinfrom which the mutant form is derived and having a coat comprisingmutant forms of the phage coat protein and no functional forms of thephage coat protein, is less infectious than a phage comprising no wildtype phage coat protein from which the mutant form is derived and havinga coat comprising at least one functional form of the phage coatprotein, and wherein the helper phage does not comprise nucleic acidencoding a functional form of the phage coat protein.

The phrase “other phage proteins” is meant to refer to proteins otherthan the functional form of the phage coat protein or the mutant form ofthe phage coat protein. Nucleic acid encoding the latter may be providedto the host cell in an alternative fashion. However, the helper phagemay further comprise nucleic acid encoding the functional form of thephage coat protein or the mutant form or both. Preferably, the helperphage does not comprise nucleic acid encoding the fusion protein. Inthis way, the helper phage is uniform and may be used to produce phagesthat preferentially comprise nucleic acid encoding the fusion protein inthe absence of nucleic acid encoding any other required helper phageprotein. Thus, preferably the fusion protein and the mutant form of thephage coat protein are encoded by separate nucleic acids. Preferably,each of the separate nucleic acids comprises a unique selection marker.Preferably, the separate nucleic acids comprise non-interfering originsof replication, wherein the origins of replication do not compete withone another resulting in bacterial cells that tolerate the separatenucleic acids for the generation of new phage particles.

The art teaches that it has been very difficult to generate helper phagebatches, wherein the helper phages harbor nucleic acid that encodes allessential proteins required for assembly of a phage particle in abacterial host cell and wherein the nucleic acid lacks a gene encodingg3p. Subsequently, the art teaches that it is difficult to produce phagelibraries using such helper phages. Several difficulties are known inthe art that hamper a proper generation of such helper phage batches.

The present invention provides methods and means and a good combinationof features such as the use of specific origins of replication,selection markers and codons in overlapping stretches of DNA, thatenable the production of phage batches containing high titers of usefulhelper phages, that can subsequently be applied for the generation ofchimaeric or infectious phages according to the invention.

Therefore, in one embodiment, the invention provides methods and meansfor the production of helper phages that carry functional forms and/orwild type forms of a phage coat protein in their coat, but thatnevertheless lack nucleic acid encoding for the functional form and/orthe wild type form of the phage coat protein. The invention provides amethod for producing a helper phage comprising the steps of: providing ahost cell with a first nucleic acid encoding a functional form of aphage coat protein; providing the host cell with a second nucleic acidencoding a mutant form of the phage coat protein, wherein the mutantform is characterized in that a phage comprising no wild type phage coatprotein from which the mutant form is derived and having a coatcomprising mutant forms of the phage coat protein and no copies of thefunctional form, is less infectious than a phage comprising no wild typephage coat protein from which the mutant form is derived and has a coatcomprising at least one functional form of the phage coat protein; andculturing the host cell to allow assembly of the helper phage. The hostcell additionally comprises nucleic acid encoding at least all otherproteins or functional equivalents thereof that are essential for theassembly of the helper phage in the host cell.

Preferably, the method does not comprise the incorporation of a wildtype form of the phage coat protein in the helper phage. Morepreferably, the phage coat protein is the g3 protein present in the coatof most, if not all bacteriophages. Preferably, the other proteins areencoded by the second nucleic acid, wherein expression of the mutantform of the phage coat protein and/or the expression of the functionalform is regulated by altering the culturing conditions of the host celland that is preferably under the control of a controllable promoter,such as the AraC/BAD, psp and lac promoter as described above.

A phage coat protein is said to be contributing significantly to theinfectiousness of a phage when it allows the phage, upon incorporationin a phage, to recognize, bind and/or infect a bacterial host in amanner comparable to a wild type version of the phage coat protein.

As used herein, the terms “less-infectious” or “non-infectious” refersto a phage, carrying no functional or wild type forms of a phage coatprotein, preferably g3p, and that exhibits a significant impaired,diminished infectious capability as compared to the wild type phage(particle) as determinable in, for instance, plaque assays well known topersons skilled in the art. As used herein, the terms “less-” and“non-infectious” may also refer to a decrease in host cell specificity.In general, a non-infectious phage is unable to recognize, bind and/orenter a bacterial cell as outlined for wild type phages in FIGS. 2 and3, whereas a less infectious phage is capable of doing so with lessefficiency.

Preferably, a phage having mutant forms infects the host cell with anefficiency which is less than 50% compared to a phage carrying at leasta functional form, more preferably less than 10%, still more preferablyless than 1%, under conditions which are otherwise comparable.

In embodiments wherein the phage coat protein comprises a g3p, themutant form preferably comprises at least a structural part, derivativeand/or analogue of the D3 region. The structural part, derivative and/oranalogue comprise at least the functionality of g3p to allow assembly ofstable phage particles.

“A derivative” of a protein as used herein comprises the same activityas that which the protein is derived from. When the protein is afunctional form of a phage coat protein, the derivative comprises thesame functionality. When the protein is a mutant form of a phage coatprotein, the derivative is also a mutant form of the phage coat protein.When the protein is a mutant form of a phage coat protein that renders aphage carrying only mutant forms of the phage coat protein lessinfectious, the derivative also is a phage coat protein that renders aphage carrying only derivatives and/or mutant forms of the phage coatprotein less infectious. Typical derivatives are proteins comprising oneor more conservative amino acid substitutions. However, derivatives mayalso comprise insertions and/or deletions. Furthermore, derivatives mayalso comprise swaps of amino acids or sequences of amino acids withinthe same protein or between two or more related and/or unrelatedproteins.

“An analogue” of a protein as used herein comprises essentially the sameactivity as the protein that the analogue is analogous to. When theprotein is a functional form of a phage coat protein, the analoguecomprises the same functionality.

Prior to the invention, it has been difficult to generate sufficientnumbers of helper phages that lack a functional g3 gene or a part,derivative and/or analogue thereof in their genome, but thatnevertheless are infectious for one round of infection through E.colicells. The present invention succeeds in generating such helper phagesefficiently. Such helper phages carry functional forms and/or wild typeg3 proteins (g3p's) on their surface, but nevertheless lack nucleic acidencoding the functional form and/or the wild type g3 protein.

The invention further provides methods and means to generate librariescomprising chimaeric phages with the help of such helper phages.Preferably, phage display libraries are generated. More preferably,libraries display a large variety of single chain Fv (scFv) fragments.Using the means and methods of the invention, phage libraries can begenerated that contain a significant higher number of infectious phagesas compared to the number of non- or less-infectious phages than weredescribed and are present in the art. At the same time the helper phagesused to produce such libraries become (through an infection round inE.coli cells) essentially non-infectious, because the g3 gene is notpresent in an infectivity-contributing form in the phages. Thus, afterinfection of a bacterial host, the phages cannot spread to otherbacterial cells except of course through division of the alreadyinfected host into daughter cells.

In another embodiment, such libraries are therefore also provided by theinvention. The generated libraries are particularly useful for panningexperiments because the titers of phages per milliliter aresignificantly higher than was used in the art until the presentinvention. Moreover, libraries of the invention display less a-specificstickiness. Thus, the libraries display less false positives thanlibraries in the art. Moreover, after one round of panning, only phagesthat display a functional form on their surface (preferably fused to aproteinaceous molecule) can be amplified in E.coli cells, while phagesthat do not carry any functional forms of the phage coat protein, butonly carry mutant forms of the phage coat protein are essentiallynon-infectious and cannot be amplified on E.coli cells. Therefore, thenumber of remaining phages that are used for a second round of panningis significantly decreased. As a result of using the chimaeric phages ofthe invention present in the libraries provided by the presentinvention, the number of panning rounds is decreased and the number ofrelevant binders is obtained in a much more sufficient manner as waspossible before the present invention.

In another aspect, the invention provides nucleic acids and helperphages comprising the nucleic acids that comprise genomic DNA sequencesin which at least the domains of g3p that are responsible andcontributing to infection are functionally removed. The invention alsoprovides helper phage genomic DNA's in which the leader and at least theD3 domain are unaffected and fused together. The nucleic acids arepreferably based on VCSM13 and M13K07 genomic sequences. Due to a lackof a functional D1 domain, phage particles produced by the nucleic acidsare essentially non-infectious.

“Essentially,” as used in this context, means that no spread or at leastsignificantly less spread of the phages to other bacterial cells thanthe production bacterium occurs through g3p provided infectiousfeatures. This absence of spread to other bacterial cells is due to theabsence of a functional form of g3p. If during production a source forwild type form or functional form of g3p is provided, produced phagescan infect a bacterium. However, if the bacterium produces phages as aresult of the infection then a resulting phage particle is not capableof infecting another bacterium unless again a source for infectious g3pis provided during production. A chimaeric phage or an infectious phageof the invention preferably comprises a part of g3p that ensures thegeneration of a stable phage particle after one round of infection inE.coli cells. To this end, the helper phage preferably comprises anucleic acid encoding a mutant form of the g3p. Preferably, the mutantform comprises D3 or a functional part, derivative and/or analoguethereof.

A phage display library, can for instance, be generated by providing acollection of bacteria with a library of nucleic acids encoding g3pfused to a range of different proteinaceous molecules and infecting thebacteria with helper phages of the invention. In a particularlypreferred embodiment, a library of phage display particles that isproduced with these helper phages contains phages that do not carry anyinfectious parts of g3p on their surface and phages that carry one ortwo full length g3p-X fusions, next to non-infectious or less-infectiousparts of g3p deletion proteins. Phages in these library mixtures that donot express g3p-X fusion proteins cannot infect bacteria anymore, sincethey were generated in the absence of infectious g3p parts that are notfused to X. X comprises a proteinaceous molecule or a fusion partner ofinterest such as immunoglobulins or fragments of immunoglobulins such asFab fragments or scFv fragments.

In one aspect, the invention provides helper phages that combine thepresence of a selection marker with the presence of a bacterial originof replication (ORI) to overcome the described problems in theproduction of g3-minus helper phages, and subsequently for thegeneration of phage display libraries. The presence of such acombination ensures the production of large amounts of helper phagesand/or helper genomes. g3-minus helper phages with an ORI and aresistance marker can be made from the helper phages VCSM13 and M13K07.These helper phages, unlike M13 or R408, do contain a kanamycinresistance gene from the Tn903 transposon and a P15A ORI that are bothinserted in the intergenic region of the phage genome.

Another aspect of the invention, is the fact that because of thisresistance gene and the presence of this particular ORI, these helperphages can grow easily in large quantities, while empty, or no plasmid-or no genome-containing bacteria are removed under the selectionpressure, and that no interferences occur between ORI's from the phagegenome and the helper plasmid or between ORI's from the phage genome andthe display library plasmids, when both nucleic acids are present in thesame bacterial host cell. VCSM13 and M13K07 contain the P15A ORI. Toprevent the disappearance of the helper genome or the helper vector, theORI's should not cause any interferences and therefore P15A derivedORI's are not used in the vector. The vector applied for the productionof helper phages is the ColE1 ORI. Besides the features and effectsmentioned above, the helper vector for the generation of helper phagesdoes not carry an F1 ORI to prevent the incorporation of the helpervector in the phage particle instead of the viral genome.

The invention also provides vectors enabling a regulated expression ofthe mutant form and/or the functional form of g3p by the use of aregulatable promoter and that furthermore contain a resistance gene thatis different from the kanamycin resistance gene present in the helpergenome. This complementary resistance is here provided by thebeta-lactamase (ampicillin) gene since its product is relatively stableand ensures complete killing of bacteria that do not express the geneproduct.

The pBAD/gIII vector (Invitrogen) can be used as a backbone vector forthe production of helper vectors of the invention. Alternatively,vectors such as pUC19 can be used. Preferably, further features of thesebasic helper vectors are that regions of sequence homology are minimizedwhich significantly decreases the possibility of homologousrecombination.

The invention further provides the use of TOP10, LMG and/or XL1 bluebacterial host cells for the production of helper phages that contain ag3-minus genome, but are nevertheless infectious due to the g3p presenton the phages because it was delivered by the helper vector. Thegenotype of the TOP10 and LMG bacteria ensures that they can transportarabinose into the cell but that they cannot metabolize it (genotype:araABCD- and araEFGH⁺). In addition, the TOP10 bacteria are recA andendA deficient which diminishes the chance of recombination andmutation. Furthermore, the TOP10 bacteria are F−, which makes themresistant to phages that might contaminate phage batches of interest.

The present invention also provides the sequence of the VCSM13 phagegenome and use thereof for the purpose of phage display libraryconstruction, cloning phage and plasmid mutants useful in generalmolecular biology methods and means.

The present invention further provides a partially deleted g3 gene thatis still present in the helper phage genome to provide stable, butessentially non-infectious helper phages that harbor infectious g3p's ontheir coat. The invention describes this partially deleted g3 gene, thatis made synthetically by using synthetic primers in such a way that thefunctionally deleted g3 gene encodes the same protein on an amino acidlevel as compared to the other part of the g3 gene that is present inthe same bacterium, but the codons that are used do not lead tohomologous recombination events. Because the leader sequences in thedifferent settings are very different, there is no need to change theseregions in the helper genome, phage display vector (with the scFvencoding genes) or helper vector. In principle, it does not matterwhether the g3 gene in the helper genome, in the phage display vector orhelper vector has been changed as long as the two overlapping (in aminoacid content) and previously homologous g3 parts that are introducedinto one E.coli cell do not match.

The present invention describes the use of codon changes in the g3 genefor the production of helper phages that are infectious due to g3p'sencoded by the helper, but that lack a wild type or at least aninfectious g3 gene in their genome and for the production of chimaericphages according to the invention. The use of codon changes ensures adiminished chance for homologous recombination effects that might occurduring the process of helper phage generation. The invention preferablyprovides the use of codon changes in the g3 gene or parts thereof forthe generation of phage display libraries in which the helper phagegenome, that is brought into an E.coli cell together with nucleic acidsencoding for g3p-X fusion proteins, is not homologous to the g3 genepresent in the DNA encoding for the g3p-X fusion protein. These codonchanges ensure that the chance for homologous recombination events inthe generation of phage display libraries is significantly decreasedthrough which the quality of these libraries and uses thereof aresignificantly improved.

Experimental Procedures

Primers

The following primers (obtained from Genset or Invitrogen) were used inthe generation of the different vectors and helper phage genomicconstructs. Most restriction enzymes hardly or fail to digest DNA iftheir corresponding palindrome is near the end of the DNA. Therefore, astretch of 8 nucleotides was added to the 5′ end of the D3, g3-minus andg3 ORF primers in which this stretch is an A/T rich non-hybridising8-mer. D3 primers D3 BamHI Forward 5′-GGATCCTCTGGTTCCGGTGATTTTGATTAT(SEQ ID NO:1) G-3′ D3 BamHI Backward5′-GGATCCAGCGGAGTGAGAATAGAAAGGAAC-3′ (SEQ ID NO:2) g3-minus primers g3minus HindIII Forward 5′ AAGCTTCTGCGTAATAAGGAGTCTTAATCAT (SEQ ID NO:3)GC-3′ g3 minus HindIII Backward 5′-AAGCTTGTTGAAAATCTCCAAAAAAAAAGC-3′(SEQ ID NO:4) g3 ORF primers g3 ORF NcoI Forward5′-CCATGGCTGAAACTGTTGAAAGTTGTTTA (SEQ ID NO:5) GC-3′ g3 ORF XbaIBackward 5′-TCTAGATTAAGACTCCTTATTACGCAGTAT (SEQ ID NO:6) G-3′ CT andN2CT primers SnaBIclon 5′-TTAGGTTGGTGCCTTCGTAG-3′ (SEQ ID NO:7) Bamlead5′-GGATCCAGCGGAGTGAGAATAGAAAGG-3′ (SEQ ID NO:8) Bg1N25′-AGATCTGGTACTAAACCTCCTGAGTACGG-3′ (SEQ ID NO:9) BamCT5′-GGATCCTCTGGTTCCGGTGATTTTGATTAT (SEQ ID NO:10) G-3′ PacIclon5′-TTGCTTCTGTAAATCGTCGC-3′ (SEQ ID NO:11) pUC-g3 primers H3leadA5′-CAAATTCTATTTCAAGGAGACAGTCATAATGA (SEQ ID NO:12)AAAAATTATTATTCGCAATTCCTTTAG-3 H3leadB5′-GATTACGCCAAGCTTGCATGCAAATTCTATTTC (SEQ ID NO:13) AAGGAGA-3′ p3endEco5′-GCTAACATACTGCGTAATAAGGAGTCTTAAGAA (SEQ ID NO:14) TTCCAGTTCTTT-3′PCR Reactions and Product Isolation

PCR reactions with D3, g3-minus and g3 ORF primers were as a standard(except for the elongation time of the DNA synthesis cycle step)performed using the following 50 μl hot start PCR scheme and theAmpliTaq PCR kit from Perkin Elmer: 1 μl 10 mM dNTP (Roche Diagnostics),4 μl 25 mM MgCl₂, 5 μl 10× PCR buffer supplied with the kit, 5 μl 2.5 μMForward primer, 5 μl 2.5 μM Backward primer, 0.3 μl 5 units/μl AmpliTaq,10-50 ng template, sterile bi-distilled water. All components were kepton ice until placing in the pre-heated PCR block. The standard programwas as follows. 12 cycles of 25 sec at 94° C., 52° C. annealing for 25sec, 72° C. polymerization ending with one cycle of 72° C. for 7 minfollowed by storage at 4° C. The time of polymerization for new helpergenome synthesis was 12 min, and for g3 amplification and for AraC geneand AraC/BAD promoter amplification this was set at 90 sec. PCRreactions with pUC-g3 primers were performed as above, but with Pwopolymerase (Boehringer Mannheim) instead of AmpliTaq, and with thefollowing program: 30 cycles of 45 sec at 94° C., 50° C. annealing for30 sec and 72° C. polymerization for 1 min, ending with one cycle of 68°C. for 8 min followed by storage at 4° C. PCR reactions with CT and N2CTprimers were performed as above, but with Taq polymerase (Gibco) insteadof AmpliTaq, and with the following program: 25 cycles of 30 sec at 96°C., 53° C. annealing for 30 sec and 72° C. polymerization for 2 min,ending with one cycle of 72° C. for 10 min followed by storage at 4° C.An exception was the PCR reaction with N2CT primers BglN2 and PacIclon,which was performed using Taq DNA polymerase recombinant (Invitrogen)with the following program: 30 cycles of 45 sec at 94° C., 55° C.annealing for 30 sec and 72° C. polymerization for 25 min, ending withone cycle of 72° C. for 10 min followed by storage at 4° C.

All PCR products were separated on 0.5%-1% TBE agarose gels containing100 ng/ml ethidium bromide. After imaging, the desired fragments werecut out using sterile disposable chirurgical knives and isolated withQiagen's gel purification kit according to the guided protocol.

Ligation Reactions

Ligation reactions were as a standard performed in the followingreaction mixtures:

-   -   50 ng vector or helper genome    -   25 ng insert    -   4 μl 5× ligation buffer (Gibco BRL)    -   1 μl T4-ligase (Gibco-BRL, 200 units/μl)    -   sterile bi-distilled water to 20 μl.

However, for the construction of the pUC-g3 helper plasmid, T4 DNAligase (Roche, 1 unit/μl) was used, while T4 DNA ligase (NEB, 400units/μl) was used in the construction of the helper phage genomes CT,N2CT and p3-minus. The mixtures were incubated overnight at 6-16° C.Then, 30 μl sterile water, 5 μl K-acetate 3M pH 4.8 acidic acid adjusted(KAc), 1 μl glycogen 10 mg/ml and 50 μl isopropanol or 96% ethanol wereadded and mixed thoroughly. After 15 min of precipitation, the tubeswere centrifuged at maximum speed at 4° C. for 10 min. The pellet wasonce washed with 1 ml 70% ethanol and after drying, dissolved in 10 μlsterile water. Half of this volume was used for electroporation togetherwith 50 μl competent cells.

Sequencing

Sequencing of the clones was performed according to the instructionguide sent along with the ABI PRISM BigDye Terminator Cycle SequencingKit (Applied Biosystems) at 50° C. annealing temperature. All cloneswere sequenced in order to verify the correctness of the products. Thesequence of the VCSM13 helper phage genome was determined by theprimer-walking method on both strands, a method generally known topersons skilled in the art. Electroporation

All bacterial strains, except those that were used for the helper phageproduction, were acquired from manufacturers as electroporationcompetent cells with the highest competence available and transformedaccording to the manufactures protocol using 0.1 cm cuvettes (BioRad).The production of helper phages, however, is dependent on TOP10 or LMGcells (Stratagene) containing the helper plasmid (pBAD/gIII-g3). Thesecells were made competent and stored at −80° C. until use as follows:One colony of the bacteria was used to inoculate in 10 ml 2×TY withampicillin (100 μg/ml) and for LMG also with tetracycline (10 μg/ml) andcultured by vigorously shaking at 30° C. overnight. Next, the cultureswere spun down at 3000 rpm for 5 min. The pellet was resuspended in 500ml fresh 2× YT including the antibiotics and cultured until OD 0.5 in a2 1 Erlenmeyer flask on a shaking platform at 37° C. These cells wereallowed to cool on ice water for 45 min and centrifuged in pre-cooledbuckets and rotor at 3000 rpm at 4° C. for 25 min in a Sorvallcentrifuge using a GLA-3000 rotor. The supernatant was discarded and thecells were slowly and carefully resuspended in 100 ml ice-cold 10%glycerol. The centrifugation and glycerol steps were repeated twice. Thefinal pellet was taken up very carefully in 5 ml 10% ice-cold glyceroland aliquoted in pre-cooled eppendorf tubes. Next, these tubes wereimmersed in a mixture of ethanol and dry ice for 5 min to ensure veryquick freezing of the cells. The tubes containing the electrocompetentcells were stored at −80° C. until use. A comparable procedure was usedto make competent cells of XL-1 (Stratagene) containing the helperplasmid pUC-g3.

Phage Production

The desired F⁺ E.coli strain is inoculated in 2× YT medium containingthe required antibiotics and cultured at 37° C. at 220 rpm until OD 0.2.The (helper-) phage is added to the culture and incubated for 30-45 minat 37° C. in a non-shaking waterbath. Then, kanamycine (50 μg/ml) isadded to the cells and cells are further incubated at 220 rpm at 37° C.for 30-45 min. Subsequently, this solution is spun at 3500 rpm at roomtemperature for 15 min. The supernatant is removed carefully and thepellet is brought to the desired volume of 2× YT medium containing allrequired antibiotics. Cells are cultured overnight at 30° C. for a phagedisplay library and at 37° C. for regular (helper-) phages on a shakingplatform.

Titer Determination

One colony of Xl1 blue (Stratagene) is inoculated in 5 ml 2×YTcontaining 10 μg tetracycline per ml (YT-T) in a 50 ml tube (Falcon) andcultured at 37° C. at 220 rpm overnight. 200 μl of this culture is addedto 5 ml YT-T and cultured until OD 0.2. Then, the phage stock is dilutedin YT and a dilution series as required is made to determine the numberof plaque forming units. For each dilution step, 100 μl of the O.D. 0.2XL1 Blue culture is taken and added to 100 μl of the phages. Thismixture is incubated for 25 min at 37° C. in a water bath (not shaking).The 200 μl of bacterial cells is pipetted onto a 2×YT-broth platecontaining the required antibiotics. The suspension is spread using asterile glass rod. After drying, the plates are inverted and transferredinto a 37° C. incubator. After overnight culture, the number of coloniesare counted. Each colony indicates the presence of 1 infective phageparticle in the original phage solution. The number of infectiousparticles per ml of the analyzed stock is calculated. The phageparticles are ELISA tested according to the protocol supplied withanti-M13 and anti-M13-HRP conjugate (Pharmacia).

Isolation of DNA from Phages

An overnight phage culture is grown as described above. If a large scaleisolation of DNA was required the BioRad Plasmid Maxi Prep kit was usedaccording to the manufactures instructions, except for the elution stepwhich is done with 10 mM Tris pH8.5 at 65° C. for 10 min. Small ormedium scale isolations were performed using Qiagen's mini-prep kitaccording to the instruction supplied with the kit except for theelution step. The elution step was performed at 65° C. for 10 min.

PEG Precipitation

The medium containing bacteria and phages is collected in 450 mlbuckets. The mixture is spun in a pre-cooled Sorvall centrifuge using aGSA-3000 rotor at 8000 rpm for 20 min. Then, 90 ml 20% PEG/2.5 M NaCl ispipetted into clean 450 ml buckets. 360 ml of the supernatant of thecentrifuged medium containing the phages is brought into the PEGcontaining buckets and mixed well. The mixture is set on ice water for 2h or overnight in the fridge. The precipitate is pelleted bycentrifugation in a pre-cooled Sorvall centrifuge at 8000 rpm for 20min. The supernatant is decanted and the buckets are left to drip outfor 5 min in order to remove as much Precipitation buffer as possible.Subsequently, 32 ml PBS/1% bovine serum albumin (BSA) is added to the450 ml buckets containing the pelleted phages and buckets are rotated ona bottle roller for 15 min. The solution is transferred to a SS-34compatible centrifuge tube and spun in a pre-cooled Sorvall centrifugecontaining a SS-34 rotor (or equivalent equipment) for 25 min at 13,000rpm. This step removes all kind of debris and small bacteria. In themeantime, the plunger is removed from a 50 ml syringe and attached to a0.45 μM filter (Whatmann). The centrifuged supernatant is transferredinto the syringe and pushed through the filter. This step removes allsmall bacteria and other cells. 8 ml 20% PEG/2.5 M NaCl is added andmixed well. The tubes are set on ice for 1 h. The high-speedcentrifugation step is repeated as described above. The supernatant isdecanted and the tube is let to drip out on a paper towel for 5 min. Thephage pellet is dissolved in 5 ml PBS/i % BSA. Then, 5 ml 100% glycerolis added to the phage solution and mixed well. Phages are stored at −20°C. Typically, the solution contains approximately 2 to 5×10¹³ infectiousphage particles per ml.

EXAMPLES

To illustrate the invention, the following examples are provided, butare not intended to limit the scope of the invention.

Example 1

Cloning of the pBAD/gIII-g3 helper vector.

The full Open Reading Frame (ORF) of the g3 gene was generated by usingM13K07 (Gibco-BRL) DNA as a template in a standard PCR reaction togetherwith g3 ORF NcoI Forward (SEQ ID NO:5) and g3 ORF XbaI Backward (SEQ IDNO:6) primers. The purified PCR product and the pBAD/gIII vector wereboth digested with NcoI (NEB) and XbaI (Roche Diagnostics)simultaneously in buffer H (Roche Diagnostics) for 4 h at 37° C. Afterligation, isolation, electroporation in TOP10 (Stratagene) and LMG(Stratagene) cells, two correct clones were selected by sequencing andgrown on a large scale followed by the isolation of the DNA. The DNA wasreprecipitated with 70% ethanol and in the presence of KAc and thepellet was washed twice with 70% ethanol. After drying, the DNA wasdissolved in sterile bi-distilled water and stored at −20° C. until use.The resulting plasmid pBAD/gIII-g3 is depicted in FIG. 5. This helpervector contains the full sized g3 gene under the control of the AraC/BADpromoter, ampicillin resistance gene and a ColE1 ORI as most importantfeatures.

Example 2

Cloning of the g3-minus helper phage genome.

VCSM13 (Stratagene) is a widely used helper phage, however, its sequenceis not publicly available. The full DNA sequence of the VCSM13 genomewas determined and is depicted schematically in FIG. 6A and innucleotide order in FIG. 6B. The use of g3 minus HindIII Forward (SEQ IDNO:3) and g3 minus HindIII Backward (SEQ ID NO:4) primers and M13K07 andVCSM13 as templates in a standard PCR reaction resulted in the formationof a PCR product that contained HindIII sites at both ends of the DNA.After separation, gel isolation and purification, digestion with HindIII(Roche Diagnostics) and re-purification of the DNA, the product wasself-ligated under standard ligation conditions and the resulting helperphage genome, named g3-minus, was electroporated into XL1 Blue cells(Stratagene).

The transformed cells were resuspended in 5 ml 2TY medium and culturedshaking at 37° C. for 1 h. Kanamycin was added to an end-concentrationof 50 μg/ml and the cells were allowed to grow at the same conditionsfor another 5 h. The culture was centrifuged at 3000 rpm for 15 min andthe supernatant passed through a 0.22 μM filter to remove bacteria. Atthe same time, a culture of exponentially growing XL-1 Blue bacteria wasprepared. Fractions of the filtrate (50-1000 μl), containing phageparticles, were added to 5 ml of XL-1 Blue bacteria and incubated at 37°C. for 30 min without shaking. The culture was centrifuged again, thesupernatant discarded and the cells were plated on 2×YT-K-T plates andtransferred into an incubator at 37° C. for overnight growth.

Eight correct clones that lack the g3 ORF (checked through the BamHIsite) and include the introduced HindIII site were isolated and used forg3-less helper phage production in the presence of the pBAD/gIII-g3helper plasmid. Two clones that were able to form phages in the presenceof the helper plasmid were kept. From these clones, a large quantity ofDNA was isolated and stored for further experiments. The obtainedVCSM13-derived g3-minus helper phage genome, in which the entire g3 geneexcept for the last six codons was replaced by a HindIII site, isdepicted schematically in FIG. 6C, while the g3-minus sequence isdepicted in FIG. 6D.

Example 3

Cloning of the D3 helper phage genome with a partially deleted g3 gene.

The construction of helper phage genome that express only the D3 part ofthe g3 gene was comparable to the above described g3-minus helper phageswith the exception that the primers used were D3 BamHI Forward (SEQ IDNO: 1) and D3 BamHI Backward (SEQ ID NO:2) primers in order to generatethe new genome. All other procedures were the same as for the g3-minusprocedure, except for the use of BamHI instead of HindIII. In the end,the DNA of two correct clones was kept and stored at −20° C. The finalVCSM13-derived construct of the helper phage genome (named D3: it onlyexpresses this part of the g3 gene that does not contribute to theinfectiousness of the phage particle) is depicted in FIG. 7A, while theD3 sequence is depicted in FIG. 7B.

Example 4

Production of infectious helper phages that do not carry a gene encodingthe wild type g3p.

The procedures for generating g3-less and generating partially deletedg3 (or D3 expressing) helper phages are identical. Frozen competentTOP10 or LMG cells that contain the pBAD/gIII-g3 helper plasmid wereelectroporated using 100 ng helper phage DNA. After recovery, the cellswere transferred into 4×250 ml 2×YT-K-A medium supplemented with 0.05%arabinose (Sigma). Phages were produced during overnight culture at 37°C. and vigorous shaking. The next day the phages were purified andstored according to the standard procedures of precipitation and storagethat were described supra. The number of infectious particles and thenumber of phages were determined by titration and ELISA procedures thatwere also described supra. For g3-less helper phages, approximately5×10¹¹ infectious particles were synthesized while for D3 approximately5×10¹³ infectious phages were formed using these procedures.

Example 5

Amplification and harvesting of phage display libraries containinginfectious phages carrying g3p-scFv fusions and non-infectious helperphages, using g3 minus and partially g3 deleted helper phages.

A frozen library was inoculated as follows. In general, approximately5-10 μl concentrated stocks were inoculated in 25 ml 2×YT containing therequired antibiotics and 5% glucose and grown at 37° C. with vigorousshaking. At OD 0.3-0.4 (after about 2-3 h), a 500-1000 fold excess ofhelper phage was added. The medium containing the helper phages andbacteria was incubated in a water bath at 37° C. without shaking for 25min. Removal of dead cells and excess of phage particles was realizedafter a centrifugation step at 3000 rpm for 15 min. The pellet wasresuspended in 250 ml 2×YT with antibiotics but without glucose andgrown at 30° C. with good aeration overnight. The next day the formedphages were isolated and stored using the standard PEG/NaCl procedure.

Example 6

Codon usage in g3 and the partially deleted g3 gene (D3) to preventhomologous recombination during helper phage production and libraryamplification.

In order to prevent possible recombinations between a genomic nucleicacid encoding the helper phage g3 protein region (or a part thereof suchas the D3 domain) and other nucleic acids (like the phage display vectorand the AraC/BAD helper vector), a series of helper phages are designedthat contain changed codons within the g3p region. Newly translatedg3p's are identical to the wild type g3 protein or protein part (D3).Due to these changes, g3-ORF coding DNA domains cannot or barelyrecombine with the phage display vectors or the AraC/BAD-g3 helpervector. The codons that are used to generate non-homologous g3 genes aredepicted in FIG. 8 and are optimal for the E.coli transcriptionmachinery. PCR generation of helper phage genomes (VCSM13, M13K07, D3,g3-minus or AraC/BAD) with g3-leader backward and g3 end forward primerswith NotI restriction sites ensure the generation of PCR productscontaining all helper phage components and genes, except for the g3 ORF.New g3 regions are constructed with overlapping primers and are insertedin helper phages. The PCR-generated g3p or parts thereof are digestedwith NotI and ligated in the NotI digested PCR generated helper phagegenome. After transformation and selection of correct helper genomes(with a new g3 gene), helper phages are grown as described.

Example 7

Selection of thyroglobulin-interacting phages using a library amplifiedwith helper phages comprising only the D3 part of g3p in their genome.

In order to validate the D3 helper phages in standard phage selections,a selection was performed using an antibody phage display library thatwas amplified using the D3 helper phages as described above. Proceduresthat were used were essentially as described by De Kruif et al. (1995a).Briefly, thyroglobulin was coated to a plastic tube. The tube wasblocked in PBS containing 2% milk (MPBS) whereafter the antibody phagedisplay library, also blocked in MPBS, was added to the tube. The phageswere allowed to bind for 2 h, whereafter non-binding phages were removedby washing the tube in PBS containing 0.1% Tween-20 as described by DeKruif et al. (1995a). The binding phages were eluted in 50 mM Glycin/HClpH 2.2 (10 min at RT) and used to infect freshly grown XL-1 Bluebacteria. The bacteria were plated on 2TY agar plates containing theappropriate antibiotics and glucose, incubated overnight at 37° C. andused to prepare an enriched phage display library; phage D3 was againused as helper phage. The procedure was repeated once, whereafterindividual E.coli colonies were used to prepare monoclonal phageantibodies. These monoclonal phage antibodies were tested in ELISA fortheir ability to bind specifically to the thyroglobulin antigen. Resultsshow that after two rounds of selection, 25/46 colonies show positivebinding to thyroglobulin. Previously we found that by using a generalVCSM-13 as a helper phage, at least 3 rounds of selection were requiredto obtain specific binders in this selection format.

Example 8

Selection of phages interacting with myeloma cells using a libraryamplified with helper phages comprising only the D3 part of g3p in theirgenome.

In order to validate the D3 helper phages in selections on intact cells,a selection was performed using an antibody phage display library thatwas amplified using the D3 helper phage. Procedures that were used wereessentially as described by De Kruif et al. (1995a and 1995b).

Briefly, myeloma cells (AML, CD33+, CD34+) were obtained from the bloodof a patient undergoing treatment at the Utrecht University Hospital(The Netherlands). 0.5 ml phage library was added to 3 ml RPMI mediumcontaining 10% FCS (RPMIS) and incubated on ice for 15 min. The myelomacells were added and the cell suspension was rotated at 4° C. for 2 h.The cells were washed 5 times in 50 ml ice-cold RPMIS whereafter thebinding phages were eluted (in 50 mM Glycin/HCl pH 2.2 for 10 min at RT)and used to infect freshly grown XL-1 Blue bacteria. The bacteria wereplated on 2TY agar plates containing the appropriate antibiotics andglucose, incubated overnight at 37° C. and used to prepare an enrichedphage display library. Again, the D3 expressing helper phages were usedas helper. The procedure was repeated once, whereafter individual E.colicolonies were used to prepare monoclonal phage antibodies. Thesemonoclonal phage antibodies were tested in FACS procedures for theirability to bind myeloma cells. Results show that 23 out of 41 clonestested bound specifically to epitopes expressed on the myeloma cells.Generally, three or more rounds of selection are required to obtainsimilar numbers of binding phages using identical procedures, with theexception of using VCSM-13 helper phages instead of the helper phagesdescribed by the invention.

Example 9

Cloning of the pUC-g3 helper plasmid.

As an alternative to the pBAD/gIII-g3 helper plasmid described above, apUC19-based helper plasmid for the expression of wild-type g3p under thecontrol of a lac promotor was constructed. The use of H3leadA andp3endEco primers and VCSM13 as a template in a PCR reaction detailedabove resulted in the formation of a PCR product containing the g3 genesequence with 28 additional nucleotides upstream of the gene and 15additional nucleotides including an introduced EcoRI site downstream.The use of H3leadB and p3endEco primers and this PCR product as atemplate in a PCR reaction detailed above resulted in the formation ofan extended PCR product containing the g3 gene sequence, with introducedHindIII and EcoRI sites upstream and downstream of the gene,respectively. The extended PCR product was cloned into plasmidpCR4-BluntII-TOPO using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen)and the sequence of the insert was checked. Plasmid pUC19 (New EnglandBiolabs) and the pCR4-BluntII-TOPO derivative containing the g3 genewere both digested with HindIII and EcoRI. The fragment containing theg3 gene from the pCR4-TOPO derivative and the vector fragment of pUC19were purified from gel and ligated. The resulting plasmid, named pUC-g3,was electroporated into XL-1 cells (Stratagene) and a correct clone wasselected by sequencing DNA isolated from the transformed cells. Helpervector pUC-g3, which contains the g3 gene under the control of the lacpromotor, is depicted schematically in FIG. 9.

Example 10

Alternative Cloning of Helper Phage Genomes with a Partially Deleted g3Gene.

In the cloning procedures described in example 2 and 3, a very largeregion of the phage genome is amplified by PCR, with the risk ofintroducing artificial mutations due to the limited fidelity of DNApolymerase. An alternative strategy was applied to produce helpergenomes with a partially deleted g3 gene.

In this strategy, a part of the g3 sequence encoding the g3 leader, anda part of the g3 sequence encoding a C-terminal portion of the g3protein, were amplified separately. The two fragments were combined in ashuttle vector and subsequently cloned into the phage genome. Todistinguish the resulting constructs from the D3 helper phage describedin example 3, the generally accepted alternative nomenclature for the g3domains was used, in which D1, D2 and D3 are named N1, N2 and CT,respectively. The use of SnaBIclon and Bamlead primers and VCSM13 as atemplate in a PCR reaction detailed above resulted in the formation of aPCR product containing the g3 leader sequence, with a native SnaBI siteupstream and an introduced BamHI site downstream. The use of BamCT andPacIclon primers and VCSM13 as a template in a PCR reaction detailedabove resulted in the formation of a PCR product containing the g3 CTdomain, with an introduced BamHI site upstream and a native PacI sitedownstream. The use of BglN2 and PacIclon primers and VCSM13 as atemplate in a PCR reaction detailed above resulted in the formation of aPCR product containing the g3 N2 and CT domains, with an introducedBglII site upstream and a native PacI site downstream.

All PCR products were cloned into plasmid pCR4-TOPO using the TOPO TACloning Kit for Sequencing (Invitrogen) and the sequences of the insertswere checked. The pCR4-TOPO derivative containing the leader sequencewas digested with NotI and BamHI, and the insert was isolated fromagarose gel. The pCR4-TOPO derivative containing the CT domain was alsodigested with NotI and BamHI, and the vector fragment was isolated. Theinsert was ligated with the vector fragment, resulting in a pCR4-TOPOderivative containing the g3 gene in which the N1 and N2 domains aredeleted. This plasmid and VCSM13 were both digested with SnaBI and PacI.After isolation, the plasmid insert was ligated with the VCSM13 vectorfragment.

The sequence of the insert in the resulting phagemid, named CT andidentical to D3 depicted in FIG. 7A, was checked. A comparable procedurewas used to construct a phagemid lacking only the N1 domain. For thisconstruct, the pCR4-TOPO derivative containing the N2 and CT domains wasdigested with NotI and BglII, and the vector fragment was isolated. Theinsert containing the leader sequence was ligated with the vectorfragment (the cohesive ends of BglII and BamHI are compatible),resulting in a pCR4-TOPO derivative containing the g3 gene in which theN1 domain is deleted. This gene was sub-cloned to VCSM13 as describedabove and the sequence of the insert in the resulting phagemid, namedN2CT, was checked. The VCSM13-derived helper phage genome N2CT isdepicted schematically in FIG. 10.

Example 11

Cloning of the p3-minus Helper Phage Genome.

In the cloning of the g3-minus helper phage genome described in example2, almost the entire g3 sequence was deleted. The g3 gene overlaps theribosome-binding site of the g6 gene and may contain other importantfeatures as well.

An alternative cloning strategy was applied, in which part of the N2domain and the entire CT domain are retained at the DNA level, but aframe shift deletes these domains at the protein level. The pCR4-TOPOderivative containing the g3 leader fused to the g3 CT domain (seeabove) and VCSM13 were both cut with BamHI and SnaBI. After isolationfrom gel, the insert fragment of the pCR4-TOPO derivative, containingthe g3 leader, was ligated with the vector fragment of VCSM13,containing a C-terminal portion of the N2 domain and the entire CTdomain of g3. The sequence of the insert in the resulting phagemid,named p3-minus, was checked. In this construct, a frame shift occurs atthe BamHI site located between the g3 leader and the C-terminal portionof the N2 domain, resulting in the introduction of over 30 stop codonsdownstream of the BamHI site. The VCSM13-derived helper phage genomep3-minus is depicted schematically in FIG. 11.

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1. A chimaeric phage having a coat comprising a mixture of proteins,said mixture of proteins comprising: a fusion protein comprising aproteinaceous molecule is fused to a functional form of a phage coatprotein; and a mutant form of said phage coat protein, wherein saidmutant form is characterized in that a phage comprising no wild typephage coat protein from which said mutant form originates and has a coatcomprising said mutant form and no copies of said functional form, isless infectious than a phage comprising no wild type phage coat proteinfrom which said mutant form originates and has a coat comprising saidmutant form and at least one copy of said functional form.
 2. Achimaeric phage having a coat comprising a mixture of proteins, saidmixture of proteins comprising: a fusion protein comprising aproteinaceous molecule is fused to a functional form of a phage coatprotein; and a mutant form of said phage coat protein, wherein saidmutant form is impaired in binding to a host cell receptor.
 3. Thechimaeric phage to of claim 1, wherein said phage coat protein is the g3protein.
 4. The chimaeric phage to of claim 3, wherein said mutant formcomprises a mutation in the D1 region, the D2 region or the D1 regionand the D2 region of said g3 protein.
 5. The chimaeric phage of claim 4,wherein said mutation comprises a deletion of substantially all of saidD1 and said D2 region of said g3 protein.
 6. The chimaeric phage ofclaim 1, further comprising a nucleic acid encoding said fusion protein.7. The chimaeric phage of claim 1, wherein said chimaeric phage is of aM13, M13K07, VCSM13 or R408 phage origin.
 8. The chimaeric phage ofclaim 1, wherein said proteinaceous molecule comprises a peptide or aprotein.
 9. The chimaeric phage of claim 1, wherein said proteinaceousmolecule is selected from the group consisting of an antibody, a Fabfragment, a single chain Fv fragment, a variable region, a CDR region,an immunoglobulin and any combination thereof.
 10. A chimaeric phagehaving a coat comprising a mixture of proteins, said mixture of proteinscomprising. a fusion protein comprising a proteinaceous molecule isfused to a phage coat protein, or to a fragment or derivative thereof;wherein said fusion protein is functional so as to render the chimaericphage infectious; and a mutant form of said phage coat protein, whereinsaid mutant form is characterized in that a phage comprising no wildtype phage coat protein from which said mutant form is originates andcarrying said mutant form and no copies of said fusion protein, is lessinfectious than a phage comprising no wild type phage coat protein fromwhich said mutant form originates and carrying in addition to saidmutant form at least one copy of said fusion protein.
 11. A chimaericphage having a coat comprising a mixture of proteins, said mixture ofproteins comprising. a fusion protein comprising a proteinaceousmolecule is fused to a phage coat protein, or to a fragment orderivative thereof; wherein said fusion protein is functional so as torender the chimaeric phage infectious; and a mutant form of said phagecoat protein, said mutant form being impaired in binding to a host cellreceptor.
 12. The chimaeric phage of claim 10, wherein said mutant formis characterized in that a phage comprising no wild type phage coatprotein from which said mutant form originates and carrying said mutantform and no copies of said fusion protein is non-infectious.
 13. Thechimaeric phage of claim 1, wherein said mutant form is furthercharacterized in that a phage having a coat comprising said mutant formin the presence or absence of copies of said functional form, is stable.14. An infectious phage comprising: at least one copy of a mutant formof a phage coat protein, wherein said mutant form has lost the abilityto mediate infection of a natural host by said infectious phage.
 15. Aphage collection comprising a the chimaeric phage of claim
 1. 16. Thephage collection of claim 15, wherein said phage collection is a phagedisplay library.
 17. A phage collection consisting essentially of thechimaeric phage of claim
 1. 18. A process for producing a phageparticle, said process comprising: providing a host cell with a firstnucleic acid encoding a fusion protein, said fusion protein comprising aproteinaceous molecule fused to a functional form of a phage coatprotein; providing said host cell with a second nucleic acid encoding amutant form of said phage coat protein, said mutant form beingcharacterized in that a phage comprising no wild type phage coat proteinfrom which said mutant form originates and having a coat comprising saidmutant form and no copies of said functional form, is less infectiousthan a phage comprising no wild type phage coat protein from which saidmutant form originates and having a coat comprising at least one copy ofsaid functional form; wherein said host cell comprises an additionalnucleic acid encoding at least all other proteins, or functionalequivalents thereof, that are essential for the assembly of said phageparticle in said host cell; and culturing said host cell to allowassembly of said phage particle.
 19. A process for producing a phageparticle, said process comprising: providing a host cell with a firstnucleic acid encoding a fusion protein, said fusion protein comprising aproteinaceous molecule fused to a functional form of a phage coatprotein; providing said host cell with a second nucleic acid encoding amutant form of said phage coat protein, said mutant form being impairedin binding to a host cell receptor; and culturing said host cell toallow assembly of said phage particle.
 20. The process according toclaim 18, wherein expression of said fusion protein, said mutant form ora combination thereof is regulatable by altering the culturingconditions of said host cell.
 21. The process according to claim 18,wherein expression of said fusion protein, said mutant form or acombination thereof is under the control of a regulatable promoter. 22.The process according to claim 21, wherein said regulatable promotercomprises the AraC/BAD promoter, the psp promoter, the lac promoter, ora functional equivalent of any thereof.
 23. The process according toclaim 18, wherein said additional nucleic acid sequence is provided by ahelper phage to said host cell.
 24. The process according to claim 23,wherein said helper phage comprises said second nucleic acid.
 25. Theprocess according to claim 18, wherein said fusion protein and saidmutant form are encoded by separate nucleic acids and each uniqueselection marker.
 26. The process according to claim 25, wherein saidseparate nucleic acids each comprises a unique origin of replication.27. The process according to claim 25, wherein said separate nucleicacids each comprises codons that essentially do not render a homologousrecombination event between said separate nucleic acids.
 28. A chimaericphase produced by the process according to claim
 18. 29. A helper phagecomprising: nucleic acid encoding phage proteins or functionalequivalents thereof that are essential for the assembly of said helperphage; wherein said nucleic acid further encodes a mutant form of aphage coat protein, said mutant form characterized in that a phagecomprising no wild type phage coat protein from which said mutant formoriginates and having a coat comprising said mutant form and no copiesof a functional form of said phage coat protein, is less infectious thana phage comprising no wild type phage coat protein from which saidmutant form originates and having a coat comprising at least one copy ofsaid functional form; wherein said functional form is characterized inthat it renders a phage particle carrying said functional form in itscoat infectious; wherein said helper phage does not comprise a nucleicacid encoding said functional form.
 30. A helper phage comprising:nucleic acid encoding phage proteins or functional equivalents thereofthat are essential for the assembly of said helper phage; wherein saidnucleic acid further encodes a mutant form of a phage coat protein, saidmutant form being impaired in binding to a host cell receptor; whereinsaid helper phage does not comprise a nucleic acid encoding a functionalform of said phage coat protein.
 31. The helper phage of claim 29,wherein said phage coat protein is the g3 protein.
 32. The helper phageof claim 31, wherein said mutant form comprises a mutation in the D1region, the D2 region, or the D1 region and the D2 region of said g3protein.
 33. The helper phage of claim 32, wherein said mutationcomprises a deletion of substantially all of said D1 and said D2 regionof said g3 protein.
 34. The helper phage of claim 29, wherein saidmutant form is further characterized in that a phage having a coatcomprising said mutant form in the presence or absence of a copy of saidfunctional form, is stable.
 35. A process for producing a helper phagethe process comprising: providing a host cell with a first nucleic acidencoding a functional form of a phage coat protein; providing said hostcell with a second nucleic acid encoding a mutant form of said phagecoat protein, wherein said mutant form is characterized in that a phagecomprising no wild type phage coat protein from which said mutant formis originates and having a coat comprising said mutant form and nocopies of said functional form, is less infectious than a phagecomprising no wild type phage coat protein from which said mutant formoriginates and having a coat comprising at least one copy of saidfunctional form; wherein said host cell comprises an additional nucleicacid sequence encoding at least all other proteins or functionalequivalents thereof that are essential for the assembly of said helperphage in said host cell; and culturing said host cell to allow assemblyof said helper phage.
 36. A process for producing a helper phage, theprocess comprising: providing a host cell with a first nucleic acidencoding a functional form of a phage coat protein; providing said hostcell with a second nucleic acid encoding a mutant form of a phage coatprotein, said mutant form being impaired in binding to a host cellreceptor; wherein said host cell comprises an additional nucleic acidsequence encoding at least all other proteins or functional equivalentsthereof that are essential for the assembly of said helper phage in saidhost cell; and culturing said host cell to allow assembly of said helperphage.
 37. The process according to claim 35, wherein said all otherproteins or functional equivalents thereof that are essential for theassembly of said helper phage in said host cell are encoded by saidsecond nucleic acid.
 38. The process according to claim 35, whereinexpression of said functional form, said mutant form or a combinationthereof is regulatable by altering the culturing conditions of said hostcell.
 39. The process according to claim 35, wherein expression of saidfunctional form, said mutant form or a combination thereof is under thecontrol of a regulatable promoter.
 40. The process according to claim39, wherein said regulatable promoter comprises the AraC/BAD promoter,the psp promoter, the lac promoter, or a functional equivalent of anythereof.
 41. The process according to claim 35, wherein said phage coatprotein is the g3 protein.
 42. The process according to claim 41,wherein said mutant form comprises a mutation in the D1 region, the D2region or the D1 region and the D2 region of said g3 protein.
 43. Theprocess according to claim 42, wherein said mutation comprises adeletion of substantially all of said D1 and said D2 region of said g3protein.
 44. The process according to claim 35, wherein said firstnucleic acid and said second nucleic acid each comprises a uniqueselection marker.
 45. The process according to claim 35, wherein saidfirst nucleic acid and said second nucleic acid each comprises a uniqueorigin of replication.
 46. The process according to claim 35, whereinsaid first nucleic acid and said second nucleic acid comprise codonsthat essentially do not render a homologous recombination event betweensaid first nucleic acid and said second nucleic acid.
 47. A helper phageproduced by the process according to claim 35, wherein said helper phagedoes not comprise nucleic acid encoding said functional form.
 48. Aprocess for the enrichment of a first binding pair member in arepertoire of first binding pair members selected from the groupconsisting of: an antibody, an antibody fragment, a single chain Fvfragment, a Fab fragment, a variable region, a CDR region, animmunoglobulin and a functional part of any thereof, wherein said firstbinding pair member is specific for a second binding pair member, theprocess comprising: contacting a the phage collection of claim 15 withmaterial comprising said second binding pair member under conditionsallowing specific binding; removing non-specific binders; and recoveringspecific binders, said specific binders comprising said first bindingpair member.
 49. The process according to claim 48, further comprising:recovering a DNA sequence encoding said first specific binding pairmember from a phase; subcloning said DNA sequence in an expressionvector; expressing said DNA sequence in a host; and culturing said hostunder conditions, wherein said first specific binding pair member isproduced.
 50. A chimaeric phage having a coat, the coat comprising: afusion protein comprising a phage coat protein fused to a proteinaceousmolecule; and a means for rendering the chimaeric phage less infectiousthan a wild-type phage from which the chimaeric phage originates. 51.The chimaeric phage of claim 50, wherein the phage coat protein is g3protein.
 52. The chimaeric phage of claim 50, wherein the proteinaceousmolecule is selected from the group consisting of an antibody, a Fabfragment, a single chain Fv fragment, a variable region, a CDR region,an immunoglobulin and any combination thereof.
 53. The chimaeric phageof claim 50, wherein the means for rendering the chimaeric phage lessinfectious comprises a mutated g3 protein.
 54. The chimaeric phage ofclaim 53, wherein the mutated g3 protein comprises a mutation in a D1region, a D2 region, or the D1 region and the D2 region.